The invention described herein was made in the performance of work under research grants from the United States Public Health Service.
Diabetes mellitus is a disease which has afflicted humans since the beginning of recorded history. Despite increased knowledge of the disease today there is still no cure for the illness. A great step forward in control of the disease was made by Banting and Best in the early 1920's when they reported successful treatment of the symptoms by injection of partially purified extracts of pancreas. These extracts were subsequently shown to contain the hormone, insulin. Today, the symptoms of the disease can be largely controlled for long periods of time by daily injection of the purified hormone.
At the present time sufficient insulin appears to be available from slaughter-house animals to supply the needs of the developed countries. Whether this will remain true when all the world's people become available for insulin therapy is questionable. The availability of insulin is closely related to the world food supply which in turn determines the amount of animal production. This may not be sufficient to supply the demands of the world population. The need for a practical synthesis of insulin is therefore obvious. Another factor influencing the need for a practical synthesis of insulin is the desirability of using a drug that is similar if not identical, to the human hormone since the animal derived insulins normally used in therapy differ in some molecular respects from human insulin. Administration of products which are not identical may elicit immunological and other side reactions which detract from the therapy. Although pork insulin is quite similar to human insulin it is not identical. Even so, there is not enough pork insulin to supply the demands of the medical profession and as a consequence the less desirable beef insulin (or mixtures of pork and beef) is the predominant commercial product.
All insulins, regardless of species of origin, are made up of two peptide chains (denoted A and B chains) which are connected together through two disulfide bridges. The human insulin molecule may be schematically represented by the following linear formula:
______________________________________ B Chain A Chain ______________________________________ 1 Phe Gly 2 Val Ile 3 Asn Val 4 Gln Glu 5 His Gln 6 7 8 9 10 11 Leu CysSS Gly Ser His Leu ##STR1## 12 Val Ser 13 Glu Leu 14 Ala Tyr 15 Leu Gln 16 Tyr Leu 17 Leu Glu 18 Val Asn Tyr 19 CysSS Cys 20 Gly Asn 21 Glu 22 Arg 23 Gly 24 Phe 25 Phe 26 Tyr 27 Thr 28 Pro 29 Lys 30 Thr Human Insulin ______________________________________
For several years now a number of scientists have developed chemical methods to synthesize the two separate A and B peptide chains of insulin. When these two synthetic chains are mixed under proper conditions, the disulfide bridges are formed to give the active hormone but in very poor yield. This poor yield at the last step in the synthesis has been a stumbling block in developing a commercial synthesis of insulin. At the present time there is sufficient information at hand to develop good and practical synthesis of the A and B peptide chains. However, synthesis of either one of the chains is a lengthy and costly procedure. The fact that 90% or more of this synthetic effort is thrown away in the last step involving the condensation of the two chains has thwarted practical synthetis of human or any other insulin.
The biosynthesis of insulin takes place through a precursor molecule proinsulin in which the end of the B-chain is connected to the beginning of the A-chain through an intermediate connecting peptide chain (C-peptide) of about 33 amino acid residues. Actually proinsulin is a single peptide chain which is converted in the cell to insulin through enzymatic splitting of the C-peptide to yield the active molecule. In proinsulin the parts of the molecule which will ultimately be the A and B chains are interconnected by disulfide bridges. The formation of these disulfide bridges takes place readily and specifically in the proinsulin molecule.
Although the animal cell finds it feasible to make insulin through the precursor, proinsulin, application of the same approach in the laboratory involves dumping about 35% of the synthetic effort at the last step. This is not attractive for an industrial synthesis process.
In greater detail, the biological synthesis of insulin occurs through a single peptide chain (proinsulin) in which the COOH-terminus of the B-chain is connected to the NH.sub.2 - terminus of the A-chain through a peptide of about 33 amino acid residues. Whereas the disulfide bonds of the proinsulin can be reduced and then reoxidized to give the parent molecule in good yield (ca. 70%), similar treatment of the two chain insulin molecule results in a poor yield of reoxidized products containing the active insulin with correct pairing of disulfide bonds. This fact has handicapped chemical syntheses of insulin involving, as a last step, the combination of the two separate chains through formation of the disulfide bonds. The three-dimensional structure of insulin has revealed that the NH.sub.2 -- terminal glycine (designated A1) of the A-chain is located quite close (ca. 10A) to the epsilon amino of lysine (designated B29) which comprises the penultimate amino acid residue of the B-chain. Recently several investigators (D. G. Lindsay, FEB LETTS., V. 21, p.105, 1972; D. Brandenburg, W. D. Busse, H. G. Gattner, H. Zahn, A. Wollmer, J. Gleimann, and W. Puls in "Peptides: 1972", H. Hanson and H. D. Jukubke, Editors, North Holland Publ. Co., Amersterdam, Holland, P.270) have prepared intramolecularly cross-linked insulins involving linkage of the amino groups Al to B29 through a series of dicarboxylic acids. In other studies, insulin derivatives which were crosslinked with suberoyl (--OC(CH.sub.2).sub.6 CO--) residues could be reduced and reoxidized to give good yields of products with the correct pairing of the disulfide bridges as judged by physical and chemical properties of the reoxidized products. (S. M. L. Robinson, I. Beetz, O. Loge, D. G. Lindsay and K. Lubke, Tetrahedron Letter. V. 12, p. 985 1973; D. Brandenburg, A. Wollmer, Hoppe-Seyler's Z. Physiol. Chem., V. 354, P. 613, 1973). However, the disadvantage of the proinsulin analogues is that the crosslinking residues cannot be removed. This disadvantage has been overcome by the use of the di(BOC) -.alpha., .alpha.'-diaminosuberoyl residue. In that process, as reported by R. Geiger, R. Obermeier, Biochem. Biophys, Res. Commun. V. 55, p. 60, 1973 and D. Brandenburg, W. Schermutzki, H. Zahn, Hoppe-Seyler's Z. Physio. Chem. V. 354, p. 1521, 1973), after removal of the BOC-groups by trifluoracetic acid, the diamino-suberoyl moiety is removed by an Edman degradation but this involves a many step process and a further complication in that the Edman degradation also removes phenylalanine B1 so that the product is des Phe B1 insulin rather than insulin.